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Prospective Blinded Study of BRAFV600E Mutation Detection in Cell-Free DNA of Patients with Systemic Histiocytic Disorders David M. Hyman1,*, Eli L Diamond2,*, Cecile Rose T. Vibat3, Latifa Hassaine3, Jason C. Poole3, Minal Patel4, Veronica R. Holley5, Goran Cabrilo5, Timothy T. Lu3, Maria E. Arcila6, Young Rock Chung7, Raajit Rampal4, Mario E. Lacouture8, Neal Rosen9, Funda Meric-Bernstam5, Jose Baselga1,7, Razelle Kurzrock10, Mark G. Erlander3, Filip Janku5,*, Omar Abdel-Wahab7,*
1Developmental Therapeutics Unit, Dept. of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY USA 10065 2Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY USA 10065 3Trovagene Inc. San Diego, CA, USA 4Leukemia Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY USA 10065 5Department of Investigational Cancer Therapeutics (Phase I Program), MD Anderson Cancer Center, Houston, TX, USA 77030 6Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY USA 10065 7Human Oncology and Pathogenesis Program, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY USA 10065 8Dermatology Service, Memorial Sloan Kettering Cancer Center, New York, NY USA 10065 9Program in Molecular Pharmacology, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY USA 10065 10Moores Cancer Center, University of California San Diego, La Jolla, CA *Equal contributors. Corresponding Author: Omar Abdel-Wahab 1275 York Ave, Box 20 New York, NY 10065 abdelwao@mskcc.org Please send reprint requests to: Omar Abdel-Wahab 1275 York Ave, Box 20 New York, NY 10065 abdelwao@mskcc.org Running Title: Assessment of Cell-free BRAFV600E DNA in the Histiocytoses Financial support for this study was provided by the Erdheim-Chester Disease Global Alliance, the Geoffrey Beene Cancer Research Center, and the Marie-Josee and Henry Kravis Center for Molecular Oncology at Memorial Sloan Kettering Cancer Center (DHM, ED, and OAW). FMB is supported by the Sheikh Khalifa Al Nahyan Ben Zayed Institute for Personalized Cancer Therapy. FJ is supported by a grant from the Sidney Kimmel Foundation. OAW is also supported by grants from the Josie Robertson Foundation and the Damon Runyon Foundation.
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Author’s Disclosure of Potential Conflicts of Interest: CRTV, LH, JCP, TLL, and MGE are all employees of Trovagene; FJ received research support from Biocartis, Transgenomic, and Trovagene.
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Abstract Patients with Langerhans Cell Histiocytosis (LCH) and Erdheim-Chester Disease (ECD) have a high frequency of BRAFV600E mutations and respond to RAF inhibitors. However, detection of mutations in tissue biopsies is particularly challenging in histiocytoses due to low tumor content and stromal contamination. We applied a droplet-digitial PCR assay for quantitative detection of the BRAFV600E mutation in plasma and urine cell-free (cf)DNA and performed a prospective, blinded study in 30 ECD/LCH patients. There was 100% concordance between tissue and urinary cfDNA genotype in treatment naïve samples. cfDNA analysis facilitated identification of previously undescribed KRASG12S mutant ECD and dynamically tracked disease burden in patients treated with a variety of therapies. These results indicate that cfDNA BRAFV600E mutational analysis in plasma and urine provides a convenient and reliable method of detecting mutational status and can serve as a non-invasive biomarker to monitor response to therapy in LCH and ECD. Statement of Significance: Patients with BRAFV600E-mutant histiocytic disorders have remarkable responses to RAF inhibition but mutation detection in tissue in these disorders is challenging. Here we identify that analysis of plasma and urinary cell-free DNA provides a reliable method to detect the BRAFV600E mutation and monitor response to therapy in these disorders.
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Introduction
Langerhans Cell Histiocytosis (LCH) and Erdheim-Chester Disease (ECD) are
heterogeneous systemic histiocytic disorders characterized by accumulation and
infiltration of histiocytes in multiple tissues of the body leading to organ compromise (1).
Although the underlying etiology of these conditions has long been enigmatic, recent
investigations have determined that both LCH and ECD are clonal disorders of myeloid-
derived precursor cells (2, 3) with a high frequency of somatic BRAFV600E mutations
(40-60% of patients) (4-7). Moreover, treatment of BRAF-mutant LCH and ECD patients
with the BRAF inhibitor vemurafenib has demonstrated dramatic efficacy revolutionizing
the care of these orphan diseases (8, 9).
The above data underline the importance of accurately identifying BRAF
mutational status in patients with systemic LCH and ECD (10, 11). Unfortunately, the
scant histiocyte content and marked stromal contamination, which are a hallmark of
these disorders, make mutation detection in tissue biopsies challenging (3, 10).
Moreover, the propensity of histiocytic lesions to involve difficult to biopsy locations such
as the brain, orbits, and right atrium frequently necessitates the use of bone biopsies
further limiting the availability of suitable tumor material for BRAF genotyping (10, 11).
Finally, the infiltrative and multifocal nature of these diseases as well as the absence of
a reliable tumor marker has made evaluation of treatment response challenging.
Given these factors, the use of circulating tumor cell-free DNA (cfDNA) to both
identify the BRAFV600E mutation and monitor response to therapy represents a
potentially transformative development for these orphan diseases. A recent pilot study of
6 patients with ECD demonstrated that BRAFV600E mutations could be detected in
cfDNA (12). However, the concordance of cfDNA BRAF mutational genotype with tissue
mutational status is not established in ECD and has never been evaluated in LCH.
Moreover, the ability of quantitative cfDNA analysis to detect dynamic changes in
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BRAFV600E mutation burden during treatment of disease has not been studied. Finally,
use of urine as a source of cfDNA for mutational detection has previously been limited to
malignancies of the genitourinary tract and offers significant advantages in sample
stability and ease of serial collection.
To evaluate the validity and clinical utility of plasma and urine cfDNA BRAF
testing in LCH and ECD patients, we performed the first of a kind blinded, prospective
multicenter study in these disorders.
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Results
Cross-sectional analysis
Data from 30 patients (25 ECD, 5 LCH) were analyzed. Patient and sample
characteristics are shown in Table 1. Of these 30 patients, initial tissue BRAFV600E
genotyping identified 15 patients to be mutant, 6 patients as wildtype, and 9 as
indeterminate. Bone represented the most common anatomic site of attempted tissue
acquisition, accounting for 36.7% of biopsies in this cohort (Table 1).
Urinary cfDNA analysis for detection of the BRAFV600E mutation was performed
on all patients and concordance between cfDNA and tissue DNA mutational results were
analyzed. There was 100% concordance between tissue and urinary cfDNA genotype in
samples from treatment naïve patients. Urinary BRAFV600E cfDNA values obtained
from any timepoint in therapy identified 16 patients as mutant and 14 as wildtype
(Supplemental Table 1) (kappa=0.88; 95% CI 0.66 to 1.0). This resulted in a sensitivity
of urinary cfDNA BRAFV600E detection of 92.9%, a specificity of 100%, a positive
predictive value of 100%, and a negative predictive value of 85.7% (all compared to
BRAFV600E detection from tissue biopsy). Overall, urinary cfDNA analysis identified 2
patients as being BRAFV600E mutant that were not known to have the BRAF mutation
previously. Subsequent tissue biopsy was performed in these patients and identified the
BRAFV600E mutation, allowing both patients to enroll in an ongoing phase II study of
vemurafenib for BRAFV600E mutant ECD and LCH patients (NCT01524978). Thus,
tissue-base genotyping resulted in 21/30 (70%) patients with definitive BRAF status
compared to 30/30 (100%) using urinary cfDNA (Figure 1A).
Urinary cfDNA analysis failed to detect the BRAFV600E mutation in 1/15 (6.7%)
patient positive by tissue biopsy. Of note, the urine and plasma utilized for cfDNA
analysis in this case were sampled while the patient was in active treatment with a BRAF
inhibitor with ongoing reduction in disease burden, whereas the tissue genotyping was
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performed prior to treatment. When considering only urinary or plasma samples obtained
from treatment naïve patients, there was a 100% concordance between tissue and
urinary cfDNA genotype from urine and plasma (Supplemental Figure 1A-B).
Plasma cfDNA and urinary cfDNA were obtained at the same time point in 19/30
(63.3%) patients. Results from plasma cfDNA for identifying the BRAFV600E mutation
were comparable to urinary cfDNA results (Supplemental Table 2 and Supplemental
Figure 1). Plasma cfDNA analysis identified 9 patients as mutant and 10 as wildtype.
BRAF genotype as determined by urinary and plasma cfDNA assay was concordant for
all samples from the 19 patients with both tests (n=26 tests), except one (which was
obtained from a patient during RAF inhibitor therapy; 96% concordance). Quantitative
BRAFV600E mutant:wildtype ratio was significantly higher in the cfDNA from plasma as
well as urine in those patients whose tissue was BRAFV600E versus wildtype (p=0.0005
and 0.002, respectively; Figure 1B-C).
Longitudinal Assessment of BRAFV600E cfDNA Burden
Comparing cfDNA BRAFV600E:BRAF wildtype ratios of pre-treatment versus
BRAF inhibitor-treated BRAFV600E mutant patients, a significant decrease in the
BRAFV600E:BRAF wildtype ratio was seen with therapy (p<0.0001; Figure 2A). Serial
samples on 13 BRAFV600E mutant patients were available, 10 of which were treated
with a BRAF inhibitor. In all patients treated with a BRAF inhibitor, serial urinary cfDNA
analysis revealed progressive decrements in the BRAFV600E allele burden (Figure 2B).
Weekly serial urinary cfDNA analysis throughout the course of BRAF inhibitor therapy
revealed that the decline in mutant cfDNA burden in response to BRAF inhibitors was
consistent with radiographic disease improvement (Figure 3A-B).
Serial cfDNA BRAFV600E burden was also assessed in 2 patients treated with
anakinra, an IL-1 receptor antagonist commonly used as an off-label treatment for ECD
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(13). Interestingly, treatment with anakinra also reduced the BRAFV600E mutant allele
burden (Figure 3C). Anakinra was subsequently discontinued in one patient and within 7
days the urinary cfDNA BRAFV600E allele burden increased. Vemurafenib was then
initiated in this patient and once again BRAFV600E allele burden as assessed in cfDNA
decreased within 2 weeks of BRAF inhibitor therapy.
In at least one patient where successful RAF inhibitor therapy was discontinued
for toxicity, urinary cfDNA BRAFV600E burden increased after vemurafenib
discontinuation which mirrored radiographic evidence of disease recurrence (Figure
3D).
Identification of a KRAS mutation in a BRAFV600 Wildtype Patient
56.7% (17/30) of the patients enrolled in this study were identified as having a
BRAFV600E mutation based on either tissue genotyping and/or cfDNA analysis. In
addition to prevalent BRAFV600E mutations in these diseases, recurrent RAS mutations
have also been recently identified in ECD (14), and therefore a non-invasive method of
diagnosing somatic mutations in BRAF wildtype ECD patients is of potential value. One
BRAF wildtype patient here was found to have a KRASG12S mutation in tissue material
taken from a cardiac ECD lesion (Supplemental Figure 2A-D). This mutation was also
found to be present by cfDNA analysis in both plasma and urine (Supplemental Figure
2E and Supplemental Table 3). Although NRAS mutations have been reported in ECD
(15), KRAS mutations have never previously been reported in these disorders.
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Discussion
This study demonstrates the utility of circulating cfDNA for reliably detecting
actionable alterations and monitoring response to therapy in histiocytic disorder patients.
We identified a high correlation of tissue mutational genotype with urine and plasma
cfDNA mutational status, establishing the utility of cfDNA mutational assessment of
BRAFV600E mutations in LCH and ECD patients. Moreover, quantitative BRAFV600E
cfDNA allele burden changed dynamically with therapy and mirrored radiographic
evaluation of disease. These findings have potentially important implications for the
initial diagnostic workup and serial monitoring of these rare disorders.
We found that 30% of patients (9/30) had an indeterminate BRAF mutation result
from tumor tissue despite concerted genotyping efforts. This high proportion of patients
with unknown tissue biopsy genotype underscores the substantial difficulty in identifying
tumor genotype information in histiocytic disorder patients. The high proportion of BRAF
genotyping test failures here likely relates to the frequent use of bone as a site of biopsy
in these disorders. Eight of the 9 (88.9%) patients with an initial unknown BRAF tissue
genotyping status had biopsies from bone. The molecular assessment of bony lesions is
challenging as morphologic assessment requires decalcification procedures that often
render the tissue unsuitable for molecular testing. Furthermore, aspirates of these
lesions often yield suboptimal material for testing, with findings of non-specific
inflammation and/or fibrosis and low histiocyte content. Of the 9 patients with
indeterminate BRAF genotype from tissue biopsy, cfDNA testing identified BRAF
mutations in 2 patients. These results have immediate therapeutic implications.
In addition to the use of cfDNA for establishing initial presence or absence of
BRAFV600E mutations, serial measurements of BRAFV600E mutant allele burden on a
variety of therapies revealed the utility of cfDNA analysis for dynamically monitoring
response to both immunomodulatory and BRAF inhibitor therapy in these disorders.
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Assessment of treatment response has been an obstacle in the treatment of adult
histiocytic disorder patients as radiographic assessments of response do not accurately
characterize the wide spectrum of anatomic sites and lesion types characteristic of these
disorders. Moreover, no formal criteria for assessment of treatment response exist for
adult LCH and ECD patients (10). Thus, these data support incorporation of urinary
and/or plasma cfDNA allele burden as a potential surrogate marker for clinical benefit in
future clinical trials and standard of care of histiocytic disorder patients. It is important to
note that the rate of decline in the BRAF mutant allele burden in urinary and plasma
cfDNA was variable between patients, underlining the need for multiple serial
assessments of allele burden following initiation of therapy. Also, given that quantitative
cfDNA BRAFV600E mutation detection mirrored response to multiple therapeutic
modalities, it is likely that cfDNA detection of BRAF mutations will serve as a good
marker of disease burden not only in response to RAF targeted therapy but also across
a range of therapeutic agents commonly utilized in these disorders.
The use of urine as the source of cfDNA as reported here particularly facilitated
routine serial monitoring of BRAFV600E allele burden. While somatic mutation detection
has been performed in cfDNA of cancer patients previously, nearly all prior studies
utilizing urinary cfDNA in cancer were restricted to patients with genitourinary
malignancies (16-18). However, urinary cfDNA detection of BRAFV600E mutations
mirrored closely the results from plasma cfDNA analysis here. Moreover, as shown in
Figure 3, urinary samples for cfDNA could be obtained on a weekly basis allowing for
disease monitoring on an outpatient basis without the need for phlebotomy or other
medical procedures. Previous studies indicate that DNA in urine can be stabilized for at
least nine days (18), whereas plasma requires processing within six hours for accurate
assessment of cfDNA (19).
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Combined use of tissue and cfDNA genotyping analyses also allowed us to
identify a KRAS mutation in a BRAF wildtype ECD patient (a mutation not previously
described in ECD). Future interrogation of RAS mutations in tumor biopsies and cfDNA
from BRAF wildtype histiocytic disorder patients may provide an additional somatic
mutational biomarker and therapy options in this patient population.
Overall, these data suggest that monitoring of BRAFV600E mutations in cfDNA
of histiocytic disorder patients provides a reliable and convenient noninvasive method to
detect BRAFV600E mutations and assess treatment response in these unique disorders.
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Patients and Methods
Patients
Between January 2013 and June 2014, 30 consecutive patients with LCH and ECD seen
at Memorial Sloan Kettering Cancer Center (MSKCC) and MD Anderson Cancer Center
(MDACC) were enrolled in the study.
Tissue biopsies were performed as part of routine clinical care, with the site of
biopsy based on radiographic and/or clinical assessment of disease involvement. 10mL
of blood and between 60-120mL of urine was collected at each time point. Plasma was
separated from blood samples using standard techniques. All samples were de-
identified, and operators performing plasma and urine cfDNA analyses were blinded to
the tissue genotype and clinical characteristics of all patients.
Institutional Review Boards at both MSKCC and MDACC approved the study
protocol.
Of note, 6 plasma and 6 urinary cfDNA values which were previously reported in
a pilot proof-of-concept study (12) were not included in the current study or data
analysis.
Tissue mutational genotyping
Initial BRAF tissue mutation testing was performed by a variety of methods as part of
routine care in CLIA-certified molecular diagnostic laboratories at MSKCC, MDACC, or
the institution from which the patient was initially referred. Tissue with a BRAFV600E
mutation identified as part of these analyses was considered positive. For tissue to be
considered negative for the BRAFV600E mutation for the purposes of this analysis, it
was required to undergo further testing by a high sensitivity assay, either Sanger
sequencing with locked nuclear acid (LNA) clamping or next-generation sequencing.
Sequencing with LNA was performed according to previously published procedures (20)
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and had a limit of detection of 0.5% mutant alleles. Massively parallel sequencing was
performed by Foundation Medicine Inc. using previously published methodologies (21)
with a minimum coverage of 500x. In patients for whom initial diagnostic tissue was
insufficient for genotyping, additional biopsies were attempted as deemed appropriate by
the treating physician. Patients were considered tumor BRAF indeterminate if they met
one of the following criteria: 1) inadequate tumor material for genotyping despite multiple
biopsy attempts, 2) declined repeated biopsy for the purpose of genotyping, 3) tissue
genotyping was ordered but no result was obtained due to failure of the tumor material to
meet technical requirements. Next-generation sequencing of genomic DNA from one
BRAF wildtype tumor tissue biopsy was performed on a panel of 30 genes (ASXL1,
CBL, CEBPA, DNMT3A, ETV6, EZH2, FLT3, HRAS, IDH1/2, JAK1/2/3, KIT, KRAS,
MPL, NPM1, NRAS, PHF6, PTEN, RUNX1, SF3B1, SH2B3, SUZ12, TET1-3, TP53,
TYK2, and WT1) by MiSeq at a depth of >500X.
Plasma and Urine cfDNA extraction and analyses
Plasma cfDNA was isolated using the QIAamp Circulating Nucleic Acid Kit (QIAGEN;
Germantown, MD) according to the manufacturer’s instructions. Urine cfDNA was
isolated as previously described (12). Urine and plasma cfDNA were quantified by a droplet digital PCR (ddPCR; QX-
100, BioRad) assay to a 44bp amplicon of RNase P, a single-copy gene as previously
described (12). Quantified DNA up to 60ng was used for mutation detection of
BRAFV600E by droplet digital PCR and KRAS mutations at codons 12 and 13 of exon 2
by massively parallel sequencing.
For BRAFV600E mutation detection, a two-step PCR assay targeting a very
short (31bp) amplicon was employed to enhance detection of rare mutant alleles in
cfDNA. The first step amplification was done with two primers flanking the BRAFV600E
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locus, where both primers contain non-complementary 5’ tags that hybridize to second
round primers. A complementary blocking oligonucleotide suppressed wildtype BRAF
amplification, achieving enrichment of the mutant BRAFV600E sequence in this step.
The second step entailed a duplex ddPCR reaction using FAM (V600E BRAF) and VIC
(wildtype BRAF) TaqMan probes to enable differentiation of mutant versus wildtype
quantification, respectively. The RainDrop ddPCR platform (RainDance; Billerica, MA)
was used for PCR droplet separation, fluorescent reading, and counting droplets
containing mutant sequence, wildtype sequence, or unreacted probe.
For each patient sample, the assay identified BRAFV600E mutation fragments
detected as a percentage of detected wildtype BRAF. As previously published,
thresholds for the BRAF assay were initially developed by evaluating a training set of
urinary cfDNA from BRAFV600E metastatic cancer patients (positives) and healthy
volunteers (negatives) using a classification tree that maximized the true positive and
true negative rates (12, 22). Using this training set, a double threshold approach with an
indeterminate range between not detected and detected was estimated yielding two
threshold values (<0.05 not detected; 0.05-0.107 indeterminate; >0.107 detected) (12).
For this current study, however, the assay was simplified to a dichotomous classifier by
combining both indeterminate and negative range as ‘not detected’ yielding a single
cutoff of ≤0.107 for not detected and >0.107 as detected. A single cutpoint was pre-
selected to evaluate the performance of this assay within this cohort for false positive
and false negative rates for the detection of BRAFV600E (this was chosen because
definitions of sensitivity and specificity are not compatible with a classifier containing an
indeterminate range).
For plasma detection, wildtype BRAF patients with metastatic cancer (13 plasma
samples) were used to determine a threshold for detection of BRAFV600E mutations.
The BRAFV600E values for this wildtype BRAF population were normally distributed and
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therefore a cutpoint equivalent to three standard deviations (0.021%) above the mean of
wildtype BRAF controls (0.031%) or >0.094% mutant to wildtype was considered
positive for BRAFV600E (12).
For KRAS mutation detection (G12A/C/D/R/S/V, G13D), a two-step PCR assay
similar to that described for BRAFV600E was employed with an initial 31 bp targeted
region, except that during the second round, flanking primers were used to add patient
specific barcodes and adaptor sequences necessary for massively parallel DNA
sequencing per manufacturer’s instructions (MiSeq, Illumina; San Diego, CA). Sequence
reads were filtered for quality (Q-score>20) and verified as matching the target sequence
(no more than 3 mismatches permitted outside the mutation region). For each sample,
KRAS mutant sequences were tallied and the percent of mutant was computed. For the
KRAS assay, the distribution of background signal in the wildtype population was
observed not to conform to a normal distribution. To be consistent with the plasma BRAF
assay approach for computing the threshold (mean + 3SD), the median and median
absolute deviation of a KRAS wildtype population was used to produce a “robust” z-
score and a cutoff of greater than 4 z-scores above the median mutant signal count of
the population (or > 0.02%) was determined to be a positive result (23). This approach is
approximately equal to the mean + 3SD threshold when the data is normally distributed
(data not shown).
Statistical Analysis
Statistical analyses were performed with GraphPad Prism V5.0 for Macintosh
(GraphPad Prism Software, San Diego, CA). The Mann-Whitney U test was used to
compare BRAFV600E mutant:wildtype ratios determined by cfDNA analysis in patients
identified as BRAF wildtype based on tissue biopsy versus those identified as
BRAFV600E mutant based on tissue biopsy. In addition, the Mann-Whitney U test was
also used to compare BRAFV600E mutant:wildtype ratios obtained from urinary cfDNA
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pre-treatment with vemurafenib versus urinary cfDNA BRAFV600E mutant:wildtype
ratios obtained following initiation of therapy with vemurafenib. Concordance of tissue,
plasma, and urinary assessment of BRAFV600E mutational detection was performed by
calculating the kappa coefficient. A two-tailed p-value <0.05 was considered statistically
significant.
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Author’s Disclosure of Potential Conflicts of Interest
CRTV, LH, JCP, TLL, and MGE are all employees of Trovagene; FJ received research
support from Biocartis, Transgenomic, and Trovagene.
Author Contributions
Conception and design: OAW, ELD, CRTV, MGE, FJ, and DMH.
Provision of study materials or patients: ELD, MP, VRH, GC, YRC, RR, JB, RK, FMB,
FJ, and DMH.
Data analysis and interpretation: OAW, ELD, CRTV, LH, JCP, TTL, MEA, MGE, FJ, and
DMH.
Manuscript writing: OAW, ELD, CRTV, JCP, TTL, MGE, FMB, FJ, and DMH.
Final approval of manuscript: All authors.
Acknowledgements
We would like to thank Saege Hancock for help with performing the KRAS assay in cell-
free DNA.
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Tables Table 1: Patient and Sample Characteristics Characteristic Number (%) Median Age (range) 56 (9-75) Sex Male Female
16 (53.3%) 14 (46.7%)
Diagnosis Erdheim-Chester Disease (ECD) 25 (83.3%) Langerhans Cell Histiocytosis (LCH) 5 (16.7%)
Sites of tissue biopsy (% of cohort (# of patients))* Bone 11 (36.7%) Abdominal soft tissue (e.g. retroperitoneum) 8 (26.7%) Skin 6 (20.0%) Central nervous system 5 (16.7%) Cardiac tissue 2 (6.7%)
Median Number organ sites involved (range) ECD LCH**
3 (0-11) 2 (1-4)
Median Number of Prior Treatments (range)*** 1 (0-4) Tissue BRAFV600E genotype
Mutant 15 (50%) Wildtype 6 (20%) Indeterminate (insufficient tissue or test failure) 9 (30%)
Median number of urine collections (per patient, range) 2 (1-8) Median number of plasma collections per patient (range)
1 (0-7)
Number of paired urine and plasma collections 27 (90%) Number of patients with initial sample acquired while off therapy
26 (86.7%)
*Several individual patients underwent more than one biopsy. **All LCH patients had multisystem disease with risk-organ involvement(11). ***Refers to number of therapies prior to first cell-free DNA analysis.
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22
Figures
Figure 1: BRAFV600E mutant allele burden in cell-free DNA (cfDNA) of urine and
plasma based on BRAFV600E tissue genotype result. (A) Pie chart representation of
BRAFV600E mutational genotype as determined by initial tissue biopsy (left) or urinary
cfDNA analysis (right). Results were recorded as BRAFV600E mutant (yellow),
BRAFV600E wildtype (white), or result indeterminate (grey). (B) Ratio of
BRAFV600E:BRAF wildtype in urinary cfDNA of patients based on BRAF mutational
status as determined from tissue biopsy (BRAFV600E mutant, BRAF wildtype, or BRAF
indeterminate). Lines and error bars for BRAFV600E mutant and BRAF wildtype patients
represents mean + standard error of the mean. (C) Ratio of BRAFV600E:BRAF wildtype
in plasma cfDNA of patients based on BRAF mutational status as determined from
tissue biopsy. Each point represents a single test result from the initial assessment of
BRAFV600E:BRAF wildtype allelic ratio in cfDNA. Dotted points represent samples
collected during therapy. The red dashed line indicates the cutpoint defining a positive
versus negative cfDNA result.
Figure 2: Effect of therapy on BRAFV600E mutant allele burden in cell-free DNA
(cfDNA) of systemic histiocytosis patients. (A) Comparison of BRAFV600E allele
burden in treatment naïve urine samples compared with urinary samples acquired
anytime during therapy. (B) Effect of RAF inhibitors on cfDNA BRAFV600E mutant allele
burden in 7 consecutive patients monitored weekly during treatment with RAF inhibitors.
The initial sample in each patient is prior to initiation of therapy. The red dashed line
indicates the cutpoint defining the presence or absence of the BRAFV600E mutation.
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23
Figure 3: Dynamic monitoring of serial urinary cell-free DNA (cfDNA) BRAFV600E
mutant allele burden in systemic histiocytosis patients. (A) Gadolinium-enhanced
T1 MRI images of ECD involvement of brain (green arrows), and 18F-FDG-PET images
of disease in the right atrium (asterisk) and testes (asterisk), pre-dabrafenib and after 2
months of dabrafenib. (B) Urinary BRAFV600E cfDNA results throughout this same
patient’s therapy. (C) Urinary BRAFV600E cfDNA results of an ECD patient treated with
anakinra followed by a period of treatment cessation and then initiation of vemurafenib.
(D) Maximal intensity projection (MIP) images of 18F-FDG-PET scan images (top)
demonstrating tibial infiltration by ECD pre-vemurafenib (left scan), following 10 weeks of
vemurafenib (middle scan), and then 16 weeks after vemurafenib discontinuation (right
scan) in an ECD patient with accompanying urinary cfDNA results for each time point
(below).
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0.01
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Figure 1
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Figure 2
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Figure 3
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Published OnlineFirst October 16, 2014.Cancer Discovery David M Hyman, Eli L. Diamond, Cecile Rose T Vibat, et al. Cell-Free DNA of Patients with Systemic Histiocytic DisordersProspective Blinded Study of BRAFV600E Mutation Detection in
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